The environmental release and ecosystem risks of illicit drugs during Glastonbury Festival.

Reported high drug use at music festivals coupled with factors such as public urination can lead to the direct release of illicit drugs into the environment. Glastonbury Festival 2019 had 203,000 attendees, its site is intercepted by the Whitelake River providing a direct route for illicit drug pollution into the local environment. We tested for popular illicit drugs such as cocaine and MDMA in the river upstream and downstream of the festival site as well as in the neighbouring Redlake River. Both rivers were sampled the weeks before, during and after the festival. Cocaine, benzoylecgonine and MDMA were found at all sample sites; concentrations, and mass loads (mass carried by the river per unit of time) were significantly higher in the Whitelake site, downstream of the festival. MDMA mass loads were 104 times greater downstream in comparison to upstream sites (1.1-61.0 mg/h vs 114.7 mg/h; p < .01). Cocaine and benzoylecgonine mass loads were also 40 times higher downstream of the festival (1.3-4.2 mg/h vs 50.4 mg/h; p < .01) (22.7-81.4 mg/h vs 854.6 mg/h; p < .01). MDMA reached its highest level during the weekend after the festival with a concentration of 322 ng/L. This concentration is deemed harmful to aquatic life using Risk Quotient assessment (RQ) and provides evidence of continuous release after the festival due to leaching of MDMA from the site. Cocaine and benzoylecgonine concentrations were not at levels deemed harmful to aquatic life according to RQ assessment yet were three times higher than MDMA concentrations. Redlake River experienced no significant changes (p > .05) in any illicit drug levels, further confirming that drug release was likely dependent on the festival site. The release of environmentally damaging levels of illicit drugs into Whitelake River during the period of Glastonbury Festival suggests an underreported potential source of environmental contamination from greenfield festival sites.

Utilization of bis-MPA Dendrimers for the Calibration of Ion Mobility Collision Cross Section Calculations.

Ion mobility-mass spectrometry (IM-MS) has become increasingly popular with the rapid expansion of available techniques and instrumentation. To enable accuracy, standardization, and repeatability of IM-MS measurements, the community requires reliable and well-defined reference materials for calibration and tuning of the equipment. To address this need, synthetic dendrimers of high chemical and structural purity were tested on three ion mobility platforms as potential calibrants. First, synthesized dendrimers were characterized by drift tube ion mobility (DTIMS), using an Agilent 6560 IM-qTOF-MS to assess their drift tube collision cross section (DTCCS) values. Then, assessment of obtained CCS values on trapped ion mobility (TIMS) and traveling wave ion mobility (TWIMS) ion mobility platforms were compared to those found by DTIMS. Across all three systems, dendrimers were found to have high potential for m/z and ion mobility calibration in the CCS range of 160-1700 Å2. To further validate their use as calibrants, drift tube calculated CCS values for dendrimers were utilized to calibrate calculations of CCS for known standards including Agilent Tuning mix, the CCS Major mix from Waters, and SPLASH LIPIDOMIX. Additionally, structures of sodiated dendrimers were computated along with theoretical CCS values which showed good agreement with the experimental CCS values. On the basis of the results presented, we recommend the use of dendrimers as alternatives and/or complementary compounds to commonly used calibrants for ion mobility platforms.

IR spectroscopy of b4 fragment ions of protonated pentapeptides in the X-H (X = C, N, O) region.

The structure of peptide fragments was studied using "action" IR spectroscopy. We report on room temperature IR spectra of b4 fragments of protonated GGGGG, AAAAA, and YGGFL in the X-H (X = C, N, O) stretching region. Experiments were performed with a tandem mass spectrometer combined with a table top tunable laser, and the multiple photon absorption process was assisted using an auxiliary high-power CO2 laser. These experiments provided well-resolved spectra with relatively narrow peaks in the X-H (X = C, N, O) stretching region for the b4 fragments of protonated GGGGG, AAAAA, and YGGFL. The 3200-3700 cm(-1) range of the first two of these spectra are rather similar, and the corresponding peaks can be assigned on the basis of the classical b ion structure that has a linear backbone terminated by the oxazolone ring at the C-terminus and ionizing proton residing on the oxazolone ring nitrogen. The spectrum of the b4 of YGGFL, on the other hand, is different from the two others and is characterized by a band observed near 3238 cm(-1). Similar band positions have recently been reported for one of the four isomers of the b4 of YGGFL studied using double resonance IR/UV technique. As proposed in this study, the IR spectrum of this ion at room temperature can also be assigned to a linear N-terminal amine protonated oxazolone structure. However, an alternative assignment could be proposed because our room temperature IR spectrum of the b4 of YGGFL nicely matches with the predicted IR absorption spectrum of a macrocyclic structure. Because not all experimental IR features are unambiguously assigned on the basis of the available literature structures, further theoretical studies will be required to fully exploit the benefits offered by IR spectroscopy in the X-H (X = C, N, O) stretching region.

Influence of a Gamma Amino Acid on the Structures and Reactivity of Peptide a(3) Ions.

Collision-induced dissociation of protonated AGabaAIG (where Gaba is gamma-amino butyric acid, NH(2)-(CH(2))(3)-COOH) leads to an unusually stable a(3) ion. Tandem mass spectrometry and theory are used here to probe the enhanced stability of this fragment, whose counterpart is not usually observed in CID of protonated peptides containing only alpha amino acids. Experiments are carried out on the unlabelled and (15)N-Ala labeled AGabaAIG (labeled separately at residue one or three) probing the b(3), a(3), a(3)-NH(3) (a(3) (*)), and b(2) fragments while theory is used to characterize the most stable b(3), a(3), and b(2) structures and the formation and dissociation of the a(3) ion. Our results indicate the AGabaA oxazolone b(3) isomer undergoes head-to-tail macrocyclization and subsequent ring opening to form the GabaAA sequence isomer while this chemistry is energetically disfavored for the AAA sequence. The AGabaA a(3) fragment also undergoes macrocyclization and rearrangement to form the rearranged imine-amide isomer while this reaction is energetically disfavored for the AAA sequence. The barriers to dissociation of the AGabaA a(3) ion via the a(3)→b(2) and a(3)→a(3)* channels are higher than the literature values reported for the AAA sequence. These two effects provide a clear explanation for the enhanced stability of the AGabaA a(3) ion.

Cyclization and rearrangement reactions of a(n) fragment ions of protonated peptides.

a(n) ions are frequently formed in collision-induced dissociation (CID) of protonated peptides in tandem mass spectrometry (MS/MS) based sequencing experiments. These ions have generally been assumed to exist as immonium derivatives (-HN(+)═CHR). Using a quadrupole ion trap mass spectrometer, MS/MS experiments have been performed and the structure of a(n) ions formed from oligoglycines was probed by infrared spectroscopy. The structure and isomerization reactions of the same ions were studied using density functional theory. Overall, theory and infrared spectroscopy provide compelling evidence that a(n) ions undergo cyclization and/or rearrangement reactions, and the resulting structure(s) observed under our experimental conditions depends on the size (n). The a(2) ion (GG sequence) undergoes cyclization to form a 5-membered ring isomer. The a(3) ion (GGG sequence) undergoes cyclization initiated by nucleophilic attack of the carbonyl oxygen of the N-terminal glycine residue on the carbon center of the C-terminal immonium group forming a 7-membered ring isomer. The barrier to this reaction is comparatively low at 10.5 kcal mol(-1), and the resulting cyclic isomer (-5.4 kcal mol(-1)) is more energetically favorable than the linear form. The a(4) ion with the GGGG sequence undergoes head-to-tail cyclization via nucleophilic attack of the N-terminal amino group on the carbon center of the C-terminal immonium ion, forming an 11-membered macroring which contains a secondary amine and three trans amide bonds. Then an intermolecular proton transfer isomerizes the initially formed secondary amine moiety (-CH(2)-NH(2)(+)-CH(2)-NH-CO-) to form a new -CH(2)-NH-CH(2)-NH(2)(+)-CO- form. This structure is readily cleaved at the -CH(2)-NH(2)(+)- bond, leading to opening of the macrocycle and formation of a rearranged linear isomer with the H(2)C═NH(+)-CH(2)- moiety at the N terminus and the -CO-NH(2) amide bond at the C terminus. This rearranged linear structure is much more energetically favorable (-14.0 kcal mol(-1)) than the initially formed imine-protonated linear a(4) ion structure. Furthermore, the barriers to these cyclization and ring-opening reactions are low (8-11 kcal mol(-1)), allowing facile formation of the rearranged linear species in the mass spectrometer. This finding is not limited to 'simple' glycine-containing systems, as evidenced by the IRMPD spectrum of the a(4) ion generated from protonated AAAAA, which shows a stronger tendency toward formation of the energetically favorable (-12.3 kcal mol(-1)) rearranged linear structure with the MeHC═NH(+)-CHMe- moiety at the N terminus and the -CO-NH(2) amide bond at the C terminus. Our results indicate that one needs to consider a complex variety of cyclization and rearrangement reactions in order to decipher the structure and fragmentation pathways of peptide a(n) ions. The implications this potentially has for peptide sequencing are also discussed.

The histidine effect. Electron transfer and capture cause different dissociations and rearrangements of histidine peptide cation-radicals.

Electron-transfer and -capture dissociations of doubly protonated peptides gave dramatically different product ions for a series of histidine-containing pentapeptides of both non-tryptic (AAHAL, AHAAL, AHADL, AHDAL) and tryptic (AAAHK, AAHAK, AHAAK, HAAAK, AAAHR, AAHAR, AHAAR, HAAAR) type. Electron transfer from gaseous Cs atoms and fluoranthene anions triggered backbone dissociations of all four N-C(alpha) bonds in the peptide ions in addition to loss of H and NH(3). Substantial fractions of charge-reduced cation-radicals did not dissociate on an experimental time scale ranging from 10(-6) to 10(-1) s. Multistage tandem mass spectrometric (MS(n)) experiments indicated that the non-dissociating cation-radicals had undergone rearrangements. These were explained as being due to proton migrations from N-terminal ammonium and COOH groups to the C-2' position of the reduced His ring, resulting in substantial radical stabilization. Ab initio calculations revealed that the charge-reduced cation-radicals can exist as low-energy zwitterionic amide pi* states which were local energy minima. These states underwent facile exothermic proton migrations to form aminoketyl radical intermediates, whereas direct N-C(alpha) bond cleavage in zwitterions was disfavored. RRKM analysis indicated that backbone N-C(alpha) bond cleavages did not occur competitively from a single charge-reduced precursor. Rather, these bond cleavages proceeded from distinct intermediates which originated from different electronic states accessed by electron transfer. In stark contrast to electron transfer, capture of a free electron by the peptide ions mainly induced radical dissociations of the charge-carrying side chains and loss of a hydrogen atom followed by standard backbone dissociations of even-electron ions. The differences in dissociation are explained by different electronic states being accessed upon electron transfer and capture.

Structure of [M + H - H(2)O](+) from protonated tetraglycine revealed by tandem mass spectrometry and IRMPD spectroscopy.

Multiple-stage tandem mass spectrometry and collision-induced dissociation were used to investigate loss of H(2)O or CH(3)OH from protonated versions of GGGX (where X = G, A, and V), GGGGG, and the methyl esters of these peptides. In addition, wavelength-selective infrared multiple photon dissociation was used to characterize the [M + H - H(2)O](+) product derived from protonated GGGG and the major MS(3) fragment, [M + H - H(2)O - 29](+) of this peak. Consistent with the earlier work [ Ballard , K. D. ; Gaskell , S. J. J. Am. Soc. Mass Spectrom. 1993 , 4 , 477 - 481 ; Reid , G. E. ; Simpson , R. J. ; O'Hair , R. A. J. Int. J. Mass Spectrom. 1999 , 190/191 , 209 -230 ], CID experiments show that [M + H - H(2)O](+) is the dominant peak generated from both protonated GGGG and protonated GGGG-OMe. This strongly suggests that the loss of the H(2)O molecule occurs from a position other than the C-terminal free acid and that the product does not correspond to formation of the b(4) ion. Subsequent CID of [M + H - H(2)O](+) supports this proposal by resulting in a major product that is 29 mass units less than the precursor ion. This is consistent with loss of HN horizontal lineCH(2) rather than loss of carbon monoxide (28 mass units), which is characteristic of oxazolone-type b(n) ions. Comparison between experimental and theoretical infrared spectra for a group of possible structures confirms that the [M + H - H(2)O](+) peak is not a substituted oxazolone but instead suggests formation of an ion that features a five-membered ring along the peptide backbone, close to the amino terminus. Additionally, transition structure calculations and comparison of theoretical and experimental spectra of the [M + H - H(2)O - 29](+) peak also support this proposal.

Proteome Cold-Shock Response in the Extremely Acidophilic Archaeon, Cuniculiplasma divulgatum.

The archaeon Cuniculiplasma divulgatum is ubiquitous in acidic environments with low-to-moderate temperatures. However, molecular mechanisms underlying its ability to thrive at lower temperatures remain unexplored. Using mass spectrometry (MS)-based proteomics, we analysed the effect of short-term (3 h) exposure to cold. The C. divulgatum genome encodes 2016 protein-coding genes, from which 819 proteins were identified in the cells grown under optimal conditions. In line with the peptidolytic lifestyle of C. divulgatum, its intracellular proteome revealed the abundance of proteases, ABC transporters and cytochrome C oxidase. From 747 quantifiable polypeptides, the levels of 582 proteins showed no change after the cold shock, whereas 104 proteins were upregulated suggesting that they might be contributing to cold adaptation. The highest increase in expression appeared in low-abundance (0.001-0.005 fmol%) proteins for polypeptides' hydrolysis (metal-dependent hydrolase), oxidation of amino acids (FAD-dependent oxidoreductase), pyrimidine biosynthesis (aspartate carbamoyltransferase regulatory chain proteins), citrate cycle (2-oxoacid ferredoxin oxidoreductase) and ATP production (V type ATP synthase). Importantly, the cold shock induced a substantial increase (6% and 9%) in expression of the most-abundant proteins, thermosome beta subunit and glutamate dehydrogenase. This study has outlined potential mechanisms of environmental fitness of Cuniculiplasma spp. allowing them to colonise acidic settings at low/moderate temperatures.

Proton-driven amide bond-cleavage pathways of gas-phase peptide ions lacking mobile protons.

The mobile proton model (Dongre, A. R., Jones, J. L., Somogyi, A. and Wysocki, V. H. J. Am. Chem. Soc. 1996, 118 , 8365-8374) of peptide fragmentation states that the ionizing protons play a critical role in the gas-phase fragmentation of protonated peptides upon collision-induced dissociation (CID). The model distinguishes two classes of peptide ions, those with or without easily mobilizable protons. For the former class mild excitation leads to proton transfer reactions which populate amide nitrogen protonation sites. This enables facile amide bond cleavage and thus the formation of b and y sequence ions. In contrast, the latter class of peptide ions contains strongly basic functionalities which sequester the ionizing protons, thereby often hindering formation of sequence ions. Here we describe the proton-driven amide bond cleavages necessary to produce b and y ions from peptide ions lacking easily mobilizable protons. We show that this important class of peptide ions fragments by different means from those with easily mobilizable protons. We present three new amide bond cleavage mechanisms which involve salt-bridge, anhydride, and imine enol intermediates, respectively. All three new mechanisms are less energetically demanding than the classical oxazolone b(n)-y(m) pathway. These mechanisms offer an explanation for the formation of b and y ions from peptide ions with sequestered ionizing protons which are routinely fragmented in large-scale proteomics experiments.

Infrared spectroscopy of fragments of protonated peptides: direct evidence for macrocyclic structures of b5 ions.

b ions are of fundamental importance in peptide sequencing using tandem mass spectrometry. These ions have generally been assumed to exist as protonated oxazolone derivatives. Recent work indicates that medium-sized b ions can rearrange by head-to-tail cyclization of the oxazolone structures generating macrocyclic protonated peptides as intermediates. Here, we show using infrared spectroscopy and density functional theory calculations that the b(5) ion of protonated G(5)R exists in the mass spectrometer as an amide oxygen protonated cyclic peptide rather than fleetingly as a transient intermediate. This assignment is supported by our DFT calculations which show this macrocyclic isomer to be energetically preferred over the open oxazolone form despite the entropic constraints the cyclic form introduces.

Carboxyl-catalyzed prototropic rearrangements in histidine peptide radicals upon electron transfer: effects of peptide sequence and conformation.

We report an unusual prototropic rearrangement in gas-phase radicals formed by collisional electron transfer from cesium atoms to protonated peptides HAL, AHL, and ALH at 50 keV. The rearrangement depends on the peptide amino acid sequence and presence or steric accessibility of a free carboxyl group. Upon electron transfer, protonated HAL and ALH rearrange to tautomers that are detected as nondissociated anions in charge-reversal mass spectra. The isomerization is minor in protonated ALH and virtually absent in HAL amide. Electron structure calculations indicate that the gas-phase ions are preferentially protonated in the His imidazole ring and consist of multiple conformers that differ in their hydrogen bonding patterns. Electron transfer to protonated HAL and AHL triggers an exothermic and dynamically barrierless transfer of the carboxyl proton onto the C-2' position of the His ring that occurs on a 120-240 ns time scale. The kinetics of this isomerization are controlled by internal rotations in the radicals to assume conformations favoring the proton transfer. The radical conformations also affect subsequent proton migrations in zwitterionic His imidazoline intermediates that reform the COOH group and result in His ring isomerization. This autocatalytic prototropic rearrangement in gas-phase peptide radicals is analogous to enzyme catalytic reactions involving His and acidic amino acid residues. In contrast to HAL and AHL, the C-2' position is sterically inaccessible in ALH radicals. These radicals undergo proton migrations to the His ring C-5' positions that have moderate energy barriers and are less efficient. RRKM calculations on the combined B3LYP and PMP2/6-311++G(2d,p) potential energy surface of the ground doublet electronic state of the peptide radicals provided rate constants that were quantitatively consistent with the dissociations observed in the gas phase. The formation of minor sequence z(1) and z(2) fragments from AHL was interpreted as occurring in the first excited state of the radical.

Infrared spectroscopy of fragments from doubly protonated tryptic peptides.

Most proteins in proteomics are identified from tandem mass spectra of doubly protonated tryptic peptides. Statistical studies indicate that these spectra fall into two distinct classes. IR spectroscopy experiments and DFT calculations performed on model b(2) ions show that peptides producing Class I spectra form protonated oxazolone ions (see figure) and not protonated diketopiperazines as proposed elsewhere.

Hidden histidine radical rearrangements upon electron transfer to gas-phase peptide ions. Experimental evidence and theoretical analysis.

Protonated peptides containing histidine or arginine residues and a free carboxyl group (His-Ala-Ile, His-Ala-Leu, Ala-His-Leu, Ala-Ala-His-Ala-Leu, His-Ala-Ala-Ala-Leu, and Arg-Ala-Ile) form stable anions upon collisional double electron transfer from Cs atoms at 50 keV kinetic energies. This unusual behavior is explained by hidden rearrangements occurring in peptide radical intermediates formed by transfer of the first electron. The rearrangements occur on a approximately 120 ns time scale determined by the radical flight time. Analysis of the conformational space for (His-Ala-Ile + H)(+) precursor cations identified two major conformer groups, 1a(+)-1m(+) and 5a(+)-5h(+) , that differed in their H-bonding patterns and were calculated to collectively account for 39% and 60%, respectively, of the gas-phase ions. One-electron reduction in 1a(+) and 5a(+) triggers exothermic hydrogen atom migration from the terminal COOH group onto the His imidazole ring, forming imidazoline radical intermediates. The intermediate from 5a is characterized by its charge and spin distribution as a novel cation radical-COO(-) salt bridge. The intermediate from 1a undergoes spontaneous isomerization by imidazoline N-H migration, re-forming the COOH group and accomplishing exothermic isomerization of the initial (3H)-imidazole radical to a (2H)-imidazole radical. An analogous unimolecular isomerization in simple imidazole and histidine radicals requires activation energies of 150 kJ mol(-1), and its occurrence in 1a and 5a is due to the promoting effect of the proximate COOH group. The rearrangement is substantially reduced in Ala-Leu-His due to an unfavorable spatial orientation of the imidazole and COOH groups and precluded in the absence of a free carboxyl group in His-Ala-Leu amide. In contrast to His-Ala-Ile and Arg-Ala-Ile, protonated Lys-Ala-Ile does not produce stable anions upon double electron transfer. The radical trapping properties of histidine residues are discussed.

Sequence-scrambling fragmentation pathways of protonated peptides.

The gas-phase structures and fragmentation pathways of the N-terminal b and a fragments of YAGFL-NH(2), AGLFY-NH(2), GFLYA-NH(2), FLYAG-NH(2), and LYAGF-NH(2) were investigated using collision-induced dissociation (CID) and detailed molecular mechanics and density functional theory (DFT) calculations. Our combined experimental and theoretical approach allows probing of the scrambling and rearrangement reactions that take place in CID of b and a ions. It is shown that low-energy CID of the b(5) fragments of the above peptides produces nearly the same dissociation patterns. Furthermore, CID of protonated cyclo-(YAGFL) generates the same fragments with nearly identical ion abundances when similar experimental conditions are applied. This suggests that rapid cyclization of the primarily linear b(5) ions takes place and that the CID spectrum is indeed determined by the fragmentation behavior of the cyclic isomer. This can open up at various amide bonds, and its fragmentation behavior can be understood only by assuming a multitude of fragmenting linear structures. Our computational results fully support this cyclization-reopening mechanism by showing that protonated cyclo-(YAGFL) is energetically favored over the linear b(5) isomers. Furthermore, the cyclization-reopening transition structures are energetically less demanding than those of conventional bond-breaking reactions, allowing fast interconversion among the cyclic and linear isomers. This chemistry can lead in principle to complete loss of sequence information upon CID, as documented for the b(5) ion of FLYAG-NH(2). CID of the a(5) ions of the above peptides produces fragment ion distributions that can be explained by assuming b-type scrambling of their parent population and a --> a*-type rearrangement pathways ( Vachet , R. W. , Bishop , B. M. , Erickson , B. W. , and Glish , G. L. J. Am. Chem. Soc. 1997, 119, 5481 ). While a ions easily undergo cyclization, the resulting macrocycle predominantly reopens to regenerate the original linear structure. Computational data indicate that the a --> a*-type rearrangement pathways of the linear a isomers involve post-cleavage proton-bound dimer intermediates in which the fragments reassociate and the originally C-terminal fragment is transferred to the N-terminus.

Structure and reactivity of a(n) and a(n) peptide fragments investigated using isotope labeling, tandem mass spectrometry, and density functional theory calculations.

Extensive (15)N labeling and multiple-stage tandem mass spectrometry were used to investigate the fragmentation pathways of the model peptide FGGFL during low-energy collision-induced-dissociation (CID) in an ion-trap mass spectrometer. Of particular interest was formation of a(4) from b(4) and a(4) (a(4)-NH(3)) from a(4) ions correspondingly, and apparent rearrangement and scrambling of peptide sequence during CID. It is suggested that the original FGGF(oxa)b(4) structure undergoes b-type scrambling to form GGFF(oxa). These two isomers fragment further by elimination of CO and (14)NH(3) or (15)NH(3) to form the corresponding a(4)and a(4) isomers, respectively. For ((15)N-F)GGFL and FGG((15)N-F)L the a(4) ion population appears as two distinct peaks separated by 1 mass unit. These two peaks could be separated and fragmented individually in subsequent CID stages to provide a useful tool for exploration of potential mechanisms along the a(4) --> a(4) pathway reported previously in the literature (Vachet et al. J. Am. Chem. Soc.1997, 119, 5481, and Cooper et al. J. Am. Soc. Mass Spectrom.2006, 17, 1654). These mechanisms result in formally the same a(4) structures but differ in the position of the expelled nitrogen atom. Detailed analysis of the observed fragmentation patterns for the separated light and heavy a(4) ion fractions of ((15)N-F)GGFL indicates that the mechanism proposed by Cooper et al. is consistent with the experimental findings, while the mechanism proposed by Vachet et al. cannot account for the labeling data. In addition, a new rearrangement pathway is presented for a(4)-CO ions that effectively transfers the former C-terminal amino acid residue to the N-terminus.

Why are a(3) ions rarely observed?

It has been determined experimentally that a(3) ions are generally not observed in the tandem mass spectroscopic (MS/MS) spectra of b(3) ions. This is in contrast to other b(n) ions, which often have the corresponding a(n) ion as the base peak in their MS/MS spectra. Although this might suggest a different structure for b(3) ions compared to that of other b(n) ions, theoretical calculations indicate the conventional oxazolone structure to be the lowest energy structure for the b(3) ion of AAAAR, as it is for other b(n) ions of this peptide. However, it has been determined theoretically that the a(3) ion is lower in energy than other a(n) ions, relative to the corresponding b ions. Furthermore, the a(3) --> b(2) transition structure (TS) is lower in energy than other a(n) --> b(n-1) TSs of AAAAR, compared with the corresponding b ions. Consequently, it is suggested that the b(3) ion does fragment to the a(3) ion, but that the a(3) ion then immediately fragments (to b(2) and a(3)) because of the excess internal energy arising from its relatively low energy and the facile a(3) --> b(2) reaction. That is why a(3) ions are not observed in the MS/MS spectra of b(3) ions.

Vibrational spectroscopy and conformational structure of protonated polyalanine peptides isolated in the gas phase.

The conformational structures of protonated polyalanine peptides, Ala(n)H(+), have been investigated in the gas phase for n = 3, 4, 5, and 7 using a combination of resonant-infrared multiphoton dissociation (R-IRMPD) spectroscopy in the NH and OH stretch regions and quantum chemical calculations. Agreement between theoretical IR and experimental R-IRMPD spectral features has enabled the assignment of specific hydrogen-bonded conformational motifs in the short protonated peptides and revealed their conformational evolution under elevated-temperature conditions, as a function of increasing chain length. The shortest peptide, Ala(3)H(+), adopts a mixture of extended and cyclic chain conformations, protonated, respectively, at a backbone carbonyl or the N-terminus. The longer peptides adopt folded, cyclic, and globular charge-solvated conformations protonated at the N-terminus, consistent with previous ion-mobility studies. The longest peptide, Ala(7)H(+), adopts a globular conformation with the N-terminus completely charge-solvated, demonstrating the emergence of "physiologically relevant" intramolecular interactions in the peptide backbone. The computed conformational relative free energies highlight the importance of entropic contributions in these peptides.

Backbone cleavages and sequential loss of carbon monoxide and ammonia from protonated AGG: a combined tandem mass spectrometry, isotope labeling, and theoretical study.

The fragmentation characteristics of protonated alanylglycylglycine, [AGG + H](+), were investigated by tandem mass spectrometry in MALDI-TOF/TOF, ion trap, and hybrid sector instruments. b(2) is the most abundant fragment ion in MALDI-TOF/TOF, ion trap, and hybrid sector metastable ion (MI) experiments, while y(2) is slightly more abundant than b(2) in collision activated dissociation (CAD) performed in the sector instrument. The A-G amide bond is cleaved on the a(1)-y(2) pathway resulting in a proton-bound dimer of GG and MeCH=NH. Depending on the fragmentation conditions employed, this dimer can then (1) be detected as [AGG + H - CO](+), (2) dissociate to produce y(2) ions, [GG + H](+), (3) dissociate to produce a(1) ions, [MeCH=NH + H](+), or (4) rearrange to expel NH(3) forming a [AGG + H - CO - NH(3)](+) ion. The activation method and the experimental timescale employed largely dictate which of, and to what extent, these processes occur. These effects are qualitatively rationalized with the help of quantum chemical and RRKM calculations. Two mechanisms for formation of the [AGG + H - CO - NH(3)](+) ion were evaluated through nitrogen-15 labeling experiments and quantum chemical calculations. A mechanism involving intermolecular nucleophilic attack and association of the GG and imine fragments followed by ammonia loss was found to be more energetically favorable than expulsion of ammonia in an S(N)2-type reaction.

Revising the proton affinity scale of the naturally occurring alpha-amino acids.

The proton affinities (PA) of the 20 naturally occurring alpha-amino acids (AA) have been determined computationally by means of density functional theory (DFT) and high-level G2(MP2) calculations. These theoretical PAs, together with data that have appeared since 1997 in the literature, are used to validate the most reasonable currently available PA scale for AAs (Harrison, A. G. Mass Spectrom. Rev. 1997, 16, 201-217.). Significant scatter is observed for the PAs of Ser, Asp, Phe, Asn, Met, Pro, Gln, Glu, Trp, His, Lys, and Arg, many of which have a basic side-chain functionality. Critical review of the available data leads to new consensus PAs for Asn, Gln, Met, and Arg of 222.4, 230.5, 223.7, and 250.2 kcal/mol, respectively.

Isotope labeling and theoretical study of the formation of a3* ions from protonated tetraglycine.

Extensive 13C, 15N, and 2H labeling of tetraglycine was used to investigate the b3+ --> a3* reaction during low-energy collision-induced dissociation (CID) in a quadrupole ion-trap mass spectrometer. The patterns observed with respect to the retention or elimination of the isotope labels demonstrate that the reaction pathway involves elimination of CO and NH3. The ammonia molecule includes 2 H atoms from amide or amino positions, and one from an alpha-carbon position. The loss of NH3 does not involve elimination of the N-terminal amino group but, instead, the N atom of the presumed oxazolone ring in the b3+ ion. The CO molecule eliminated is the carbonyl group of the same oxazolone ring, and the alpha-carbon H atom is transferred from the amino acid adjacent to the oxazolone ring. Quantum chemical calculations indicate a multistep reaction cascade involving CO loss on the b3 --> a3 pathway and loss of NH=CH2 from the a3 ion to form b2. In the postreaction complex of b2 and NH=CH2, the latter can be attacked by the N-terminal amino group of the former. The product of this attack, an isomerized a3 ion, can eliminate NH3 from its N-terminus to form a3*. Calculations suggest that the ammonia and a3* species can form various ion-molecule complexes, and NH3 can initiate relay-type mobilization of the oxazolone H atoms from alpha-carbon positions to form a new oxazolone isomer. This multiple-step reaction scheme clearly explains the isotope labeling results, including unexpected scrambling of H atoms from alpha-carbon positions.